Date of Submission
5-2024
Document Type
Thesis
Degree Name
Master of Science in Forensic Science
Department
Forensic Science
Advisor
Robert H. Powers, Ph.D.
Committee Member
Virginia M. Maxwell, Ph.D.
Committee Member
Christina Zito, Ph.D.
Keywords
Ethanol, Gamma-hydroxybutyric Acid (GABA), Central Nervous System (CNS) Depressant, Alcohol Consumption Effects
MeSH
Ethanol, gamma-Aminobutyric Acid, Central Nervous System Depressants
LCSH
Ethanol, GABA, Central nervous system depressants, Drinking of alcoholic beverages
Abstract
Ethanol is the primary active ingredient in alcoholic beverages, and functions in the body as a central nervous system (CNS) depressant. The overt effects ethanol has on the body, e.g. diminished cognitive ability, loss of motor control, slurred speech, impaired balance, etc., are well documented. While the behavioral consequences of consumption are well understood, how ethanol actually functions on a mechanistic level to elicit those effects has yet to be satisfactorily elucidated.
The structural homology between ethanol and the C3 and C4 carbons of gamma-hydroxybutyric acid (GABA), the major inhibitory neurotransmitter in the mature CNS, has led us to hypothesize that the mechanism by which ethanol causes CNS depressant effects may be related in part, to the inhibition of GABA catabolic enzymes. This inhibition would be expected to increase the functional concentration of GABA in the inhibitory neuronal pre-synaptic neurotransmitter vesicles, and the inter-neuronal synaptic space following a depolarization event.
The two primary enzymes involved in GABA catabolism are GABA-Transaminase (GABA-T) and Succinyl Semialdehyde Dehydrogenase (SSADH). GABA-T exchanges an amine group from GABA with the keto group from the co-substrate a.-ketoglutarate to produce succinyl semialdehyde (SSA) and glutamate. SSA is subsequently oxidized to succinic acid via SSADH, with either NAD+ or NADP+ as the oxidizing agent. Inhibition of either, or both, enzymes would slow the catabolism of GABA, resulting in increased intracellular concentration. We have evaluated aspects of this hypothesis using a combined GABA-TIS SADH mix, and have demonstrated an overall effect of ethanol on the biphasic enzyme system. This research is focused on an effort to distinguish the effects of ethanol, if any, on each enzyme in the linked system.
To test this hypothesis, we modified the enzyme assay protocol outlined in Tsukatani, Higuchi & Matsumoto (2005). Activity of the enzyme system was measured indirectly by the amount of NADH produced using a UV-Vis spectrophotometer @ 340 nm. A series of substrate-velocity experiments were conducted with varying concentrations of GABA to evaluate the ethanol-based inhibition of the enzyme system. To isolate the effect on GABA-T, we utilized a pre-incubation period where the necessary cofactor for the oxidation by SSADH, NAD+, was initially withheld. After the pre-incubation, NAD+ was added to the reaction and the absorbance was monitored. As such, if and to the extent that ethanol inhibited the GABA-T reaction, the concentration of SSA available as a substrate for the subsequent NAD+ dependent reaction would be reduced.
Michaelis-Menten coefficients (KM) and maximal reaction rates (VMAX) were then determined to evaluate the type inhibition recorded. VMAX was unaffected by the addition of ethanol. KM of the enzyme system as a whole, and specifically for GABA-T, was increased upon the addition of ethanol. These findings are characteristic of competitive inhibition. Therefore, at physiologically relevant levels, ( e.g. 0.1, 0.2 and 0.37 g/dL) ethanol acts as a competitive inhibitor of GABA-T, which can explain the behavioral effects seen with alcohol consumption.
Recommended Citation
Corrigan, Mary, "Effects of Ethanol on Gaba Transaminase" (2024). Master's Theses. 223.
https://digitalcommons.newhaven.edu/masterstheses/223